Composition containing a complex of a polyvalent metal and a DNA-sodium salt

ABSTRACT

Compositions are provided which contain a complex of a polyvalent metal and sodium salt, the DNA-sodium salt being obtained from sturgeon milt. The complexes exhibit high antiviral activity and low toxicity.

This is a division of application Ser. No. 08/696,623, filed Aug. 14,1996, now U.S. Pat. No. 5,662,889 which is a divisional of applicationSer. No. 08/016,014, filed Feb. 10, 1993, now U.S. Pat. No. 5,547,684.

FIELD OF THE INVENTION

This invention is directed to pharmaceutical compositions containingDNA-sodium salts which stimulate the productions of white blood cells,lymphocytes and neutrophils in the peripheral blood system and thereforeare useful in the treatment of various diseases. In one aspect, theinvention is directed to certain metal complexes of DNA-sodium saltshaving antiviral activity. In another aspect the invention relates tocosmetic formulations containing DNA-sodium salts. In a further aspect,the present invention relates to an improved method of obtainingDNA-sodium salts from natural sources.

BACKGROUND OF THE INVENTION

Over the years, a wide variety of compounds which exhibit desirablebiological properties have been obtained from plants and animal sources.Upon identification of the chemical structure of the activeingredient(s), it was possible, in many cases, to synthesize the activecomponent using known chemical techniques and therefore prepare thecompounds in large quantities and at reasonable costs.

More recently, in a search for new bioactive materials, investigationshave been directed to the components of the nucleus of cellularstructures and methods for extracting and refining biologically activematerials contained therein. Various studies have been reported in theliterature of the extraction and refinement of genetic materialscontained in both plants and animals, and a wide variety ofmicroorganisms. Methods have been developed for extracting geneticmaterials from cells for further study and modifications through geneticengineering techniques.

Medical researchers, biologists, chemists and other scientists arecontinually searching for, and evaluating new sources of materials inthe hope of finding compounds which possess unexpected and usefulproperties which might make them useful in the treatment of variousdiseases. The present invention is therefore directed to the extraction,refinement and use of biological materials possessing most desirablecharacteristics for use in the pharmaceutical field.

Prior to the present invention, it had been known that a high polymerDNA could be obtained from sturgeon soft roe by extraction of the DNAwith sodium pyrophosphate. Additionally, methods have been disclosed forobtaining a sterile sodium DNA salt from aquatic animal sources.

The invention is therefore directed to an improved method by which DNAproducts could be obtained in relatively high yields from aquaticanimals, such as sturgeon by a process which avoids the use of excessiveamounts of solvent such as ethanol. Additionally, it was desired toprovide new DNA sodium salts having improved properties and greaterpotency than the DNA sodium salt obtained by prior art methods.

Accordingly, one or more of the following objects will be achieved bythe practice of this invention. It is an object of this invention toprovide a composition in biologically pure form, which is capable ofstimulating the production of white blood cells in a warm-bloodedanimal. Another object of the invention is to provide a compositionwhich can also stimulate the production of lymphocytes and neutrophilsin the peripheral blood system of a warm-blooded animal. A furtherobject is to provide a novel composition which is useful in thetreatment of various diseases. Another object is to provide certainmetal complexes of the composition which have antiviral activity. Afurther object is to provide cosmetic formulations which containDNA-sodium salts. A still further object is to provide an improvedprocess for the extraction of a native DNA salt from animal cells inrelatively high yields and without the use of large quantities ofsolvents. These and other objects will readily become apparent to thoseskilled in the art in light of the teachings herein set forth.

SUMMARY OF THE INVENTION

The present invention relates in general to novel, highly pureDNA-sodium salts which stimulate the production of white blood cells,lymphocytes and neutrophils and their use in the treatment of a varietyof diseases. The DNA-sodium salts are also useful in cosmeticformulations. The invention is also directed to certain metal complexesof these DNA salts and to an improved method for the production ofbioactive materials, and particularly to the preparation of novelDNA-sodium salts.

The DNA-sodium salt products of the present invention, hereinafter alsoreferred to as "NPD" can also be used in the form a pharmaceuticalcompositions for the treatment of (a) the side effects from theadministration of chemo and/or radiation therapy; (b) irregularities ofthe surfaces of various organs (i.e., throat, stomach, intestine and thelike); (c) vascular diseases such as phlebitis and hemorrhoids; (d)diabetes mellitus; (e) burns, particularly those due to overexposure toradiation; (f) external wounds; and (g) chronic viral conditionsincluding those caused by the HIV and related viruses. The compositionscan be administered in a variety of forms including intranasal,intramuscular and as topical compositions. In addition, the compositionsof the present invention can be used as a cosmetic especially or therejuvenation of aging skin.

DETAILED DESCRIPTION OF THE INVENTION

The DNA-sodium salts of the present invention are obtained fromnaturally-occurring sources such as animal sources, and in particularfrom the reproduction cells of certain aquatic animals such as sturgeonmilt. Although the milt from seruga or beluga can also be used, in othertypes of fish, the DNA structure and compositions are sufficientlydifferent so as to render them undesirable for the same utilitiesdescribed above.

The active ingredient of the compositions can be prepared by a processcomprising:

(1) homogenizing the sturgeon milt in an aqueous solution of sodiumchloride and sodium citrate to provide a homogenized material;

(2) washing said homogenized material with an aqueous solution of sodiumchloride and sodium citrate;

(3) contacting said washed material in a reaction chamber with:

(a) an aqueous solution of sodium chloride and sodium citrate,

(b) a detergent, and

(c) an aqueous sodium chloride solution;

(4) separating and concentrating a liquid fraction containing thebioactive material;

(5) washing said concentrated liquid fraction until a negative proteinreaction is indicated in the permeate; and

(6) thereafter, recovering a refined bioactive material as theDNA-sodium salt.

As indicated, the process is generally conducted beginning with thehomogenization of the milt material after it has been obtained from thefish and cleansed. Homogenization is conducted in an aqueous solution ofsodium chloride and sodium citrate. In practice, the solution containsfrom about 0.01 to about 0.8 M, preferably from about 0.05 M to about0.2 M of sodium chloride and from about 0.005 M to about 0.05 M ofsodium citrate. From about 10 grams to about 150 grams of the startingmaterial (milt) will be homogenized in about 20 to about 2000milliliters of the aqueous solution. Homogenization is effected fromabout 3 seconds to about 20 minutes at a temperature of from about 4° C.to about 40° C.

In the second step, the homogenized material is washed from about 1 toabout 7 times with the same volume of sodium chloride-sodium citratesolution. Thereafter, in the third step, the washed material is placedin an enamel reaction chamber equipped with a heating/cooling jacket anda rotor mixer, and containing the following additional components:

(a) from about 0.01 M to about 0.9 M of the sodium chloride-sodiumcitrate solution,

(b) from about 0.1% to about 6% by weight of a detergent, and

(c) from about 1 M to about 3 M of an aqueous sodium chloride solution.

Cell and nuclear membrane lysis, protein separation from DNA, proteindenaturing and protein-sediment formation take place in the reactionchamber in the third step.

The temperature in the chamber will usually be within the range of fromabout 10 to about 80° C., and the reaction time will usually be fromabout 3 to about 24 hours.

In the fourth step, the liquid fraction of the bioactive material, theDNA-sodium salt, is separation from the reaction mass by centrifugatingor by filtration. The liquid fraction can, if desired, be processed byultrasound at about a 22 kHz frequency for about 15-20 minutes at anintensity of about 10-12 W/cm. Finally, after the first and sixth steps,the material is further washed until a negative protein reaction isindicated and the DNA-salt recovered.

The bioactive DNA-salt material obtained by the above process ischaracterized by the following properties. It is a white powdercontaining not less than about 80% by weight of native DNA-sodium saltextracted from the sturgeon milt, not more than about 1% by weight ofproteins, not more than about 17% by weight moisture, and the remaindersodium chloride.

The nucleotide composition of the DNA extracted from sturgeon milt is asfollows:

    ______________________________________                                               Nucleotide                                                                           Mole Percent                                                    ______________________________________                                               Adenine                                                                              29.0 ± 0.9                                                          Thymine                                                                              27.0 ± 0.9                                                          Guanine                                                                              22.0 ± 0.9                                                          Cytosine                                                                             20.0 ± 0.9                                                   ______________________________________                                    

The native DNA sodium salt has a molecular weight of about 270-500×10³daltons and a hyperchromicity effect (G): G 37%.

Hyprochromicity is determined from the difference in absorption spectrumof denatured and native preparation solutions at wave length of 260 nm.

0.15 g of the preparation (precise measurement) is placed into ameasured 25 ml retorts 0.1% sodium chloride solution is added, thepreparation is dissolved and the volume is brought up to the mark(Solution 1). 1 ml of the received Solution 1 is placed into a measured200 ml retort, the volume is brought up to the mark with 0.1% sodiumchloride solution. Absorption of the received solution (D₂₆₀) ismeasured.

5 ml of Solution 1 is placed into a retort with thin section, 5 ml of10% chloric acid solution, closed and boiled on a water bath for 20 min.The solution is cooled own to the normal temperature, transferred byquantity into a measured 100 ml retort, the volume is brought up to themark with water. 5 ml of this solution is placed into a measured 50 mlretort and the solution volume is brought up to the mark with water.Absorption of the received solution (D¹ ₂₆₀ ) is measured.

Hypochromicity is calculated according to the following formula:##EQU1## H%--hypochromicity in %, D₂₆₀ --native preparation solutionabsorption,

D¹ ₂₆₀ --denatured preparation solution absorption.

As indicated above, the process for preparing a DNA-sodium salt utilizesa detergent in step (3). Preferred are the sodium slat detergents andinclude sodium dodecyl laurylsulfate, sodium xilosulfonate, sodiumacetotri-hydrate, sodium benzoate and the like.

Sodium salts of other strong acids can be used instead of NaCl, forexample, sodium sulphate, sodium phosphate, sodium phenylphosphate, andthe like.

Sodium salts of other weak acids can be used instead of sodium citrate,for example, sodium acetate, ethylenediaminetetraacetic acid disodiumsalt and the like.

In other embodiments as hereinafter indicated, the DNA sodium saltobtain by the aforementioned process can also be complexed with certainwater-soluble metal salts or it can be used in cosmetic formulations.

The DNA-sodium salts obtained in accordance with the present inventioncan be formulated into pharmaceutical compositions comprised of theactive component and one or more suitable pharmaceutically acceptablecarriers. Although a variety of pharmaceutical compositions can beprepared, it has been found that only solutions of sodium chloride canbe used for dissolving the DNA. Other salts were observed to alter thecharacteristics of the compositions.

In practice, pharmaceutical compositions prepared in accordance with thepresent invention will have a concentration of sodium chloride withinthe range of from about 0.1 percent to about 0.9 percent by weight. Atsalt concentration below about 0.1 percent, the native DNA sodium saltis destroyed while at concentrations higher than about 0.9 percent byweight, it is not only difficult to filter the DNA-containing solutionbut treatment can cause complications.

Although pharmaceutical compositions can be prepared containingdifferent concentrations of native DNA sodium salts, three typicalformulations have been prepared and evaluated. The formulations have thefollowing designations and composition by weight.

    ______________________________________                                        NPD-A    Sterile 1.5 percent solution of NPD in a 0.1 percent                          aqueous solution of NaCl and stored in 5 ml aliquots.                NPD-B    Sterile 0.25 percent solution of NPD in a 0.1 percent                         aqueous solution of NaCl and stored in 10 and 20 ml                           aliquots.                                                            NPD-C    Sterile 0.25 percent solution of NPD in a 0.1 percent                         aqueous solution of NaCl and stored in 40 ml                                  aliquots.                                                            ______________________________________                                    

The concentration of the native DNA sodium salt in the pharmaceuticalcompositions can range up to about 4 percent by weight. However, at thisconcentration, the solutions become very viscous and almostgel-like--the preferred concentration is from about 0.1 to about 4percent by weight based on the total weight of the pharmaceuticalcomposition for formulations NPD-B and NPD-C and from about 0.2 to about4 percent for formulation NDP-A. Particularly preferred compositionscontain from about 0.1 to about 2.5 percent.

If desired, other ingredients can be included with the DNA sodium saltsin the pharmaceutical compositions of this invention. Both inert andactive ingredients can be employed as long as they do not adverselyreact with or adversely effect the main active component.

The pharmaceutical preparations of the present invention can beformulated for administering the active component to a patient byinjection, intranasally or topically in a suitable cream typecomposition.

Injectable NPD-A and intranasal NDP-B are useful for the treatment ofside effects of chemotherapy and radiation therapy. An injection of 5 mlof the NPD-A formulation is given within 2-15 days after therapy.Alternatively, 5 ml portions of NPD-B may be used as a mouth cavitygargle 6 to 8 times a day for 3 to 4 days.

For the treatment of vascular diseases, only the intranasal formulationis used. 2 drops of NPD-B are placed into each nostril 4-10 times a dayfor 30 days.

The following examples are illustrative of this embodiment of theinvention:

EXAMPLE 1

1 kg of frozen sturgeon milt is cut to fragments and homogenized in 2liter of citrate-salt solution consisting of 0.15 M of sodium chlorideand 0.0021 M of sodium citrate at normal temperature. The obtainedhomogenate is then poured into the reactor, in which 34 liters ofcitrate-salt solution is already found. Then 4 liters of 6% sodiumdodecylsulphate solution in 45% solution of pure ethanol (240 grams in 4liters of 45% ethanol). The obtained mass is warmed up to 60-65° C.within 1-1.5 hours and stirred by a rotor mixer for 1.5 hours. Then 40liters of 5 M NaCl solution are added and the mixture is stirred foranother 1.5 hours. Then the resulting mass is cooled to 12-16° C. andprocessed by ultrasound at the frequency of 22 kHz for 45 minutes withthe intensity of 10-12 W/cm². The liquid fraction is concentrated byultrafiltration method in columns with hollow fibers, NaCl, water andwater-soluble low polymer protein go to the permeate, DNA-Na remains inthe liquid fraction which is then concentrated up to 6% of DNA-Na, thatis 6 or 7 times. After that the concentrate is washed with the help ofdiafiltration until negative reaction for water-soluble protein isobtained in the permeate. As a result, approximately 10-12 liters ofnative DNA-Na sodium salt 6-7% solution are obtained, with the DNA-Napossessing the following characteristics:

    ______________________________________                                        Molecular weight:                                                                              0.3-0.5 MD;                                                  Protein:         not more than 1.0% by weight                                 Hypochromicity:  not less than 87%                                            ______________________________________                                    

The concentrate containing 6-7 % of native DNA sodium salt is processedby 96% rectified ethanol (1:1). The sediment is separated and washed,first by 70% ethanol aqueous solution from NaCl, then by 96% ethanolfrom moisture, then dried in a special drying chamber at the temperatureof 40° C.

EXAMPLE 2 Preparation of an Injectable Composition Containing NPD

An injectable formulation containing the DNA sodium salt of the presentinvention was prepared by dissolving 1 gram of sodium chloride in 1liter of bidistilled non-pyrogenic water at room temperature (25° C.).311.25 grams of NPD powder were added to the solution and dissolved bystirring for 3 hours at room temperature. The resulting solution wasfiltered through a system of consecutive membrane filters of 0.8microns, 0.65 microns, 0.45 microncs and 0.22 microns. The filtrateobtained from the last filter was transferred into sterile, glasscontaining in 10 or 20 ml aliquots, closed, sealed and labelled.

EXAMPLE 3 Preparation of an Intranasal Composition Containing NPD

A pharmaceutical composition which is useful for the intranasaladministration of the DNA sodium salt to a patient is prepared bydissolving 1 gram of sodium chloride in 1 liter of bidistillednon-pyrogenic water at room temperature (25° C.). 31.25 grams of NPDpowder were added to the solution and dissolved by stirring for 1.5hours at room temperature. The resulting solution was filtered through asystem of consecutive membrane filters of 0.8 microns, 0.65 microns,0.45 microns and 0.22 microns. The filtrate obtained from the lastfilter was transferred into sterile, glass containers in 10 or 20 mlaliquots, closed, sealed and labelled.

In the following example, the pharmaceutical compositions of the presentinvention were used for the treatment of side effects of chemotherapyand radiation therapy in male and female children ranging in age from 5to 15 years and afflicted with tumors. Treatment was administered at theNational Oncology Center of the USSR Academy of Medical Sciences andinvolved chemotherapy or radiation therapy. Blood analysis beforetherapy, after therapy and after treatment with the composition of thisinvention are set forth. In many cases, the patient had received earliertreatment at their local hospital for their conditions before beingadmitted to the National Oncology Center.

EXAMPLE 4

A female child, weight 20 kg, height 122 cm, 7 years old, was diagnosedas having left nephroblastoma.

Treatment

Aug. 16, 1991-Aug. 20, 1991: an aggressive polychemotherapy course wasadministered:

    ______________________________________                                        1.       vepesid        i/v by 80 mg N5                                       2.       cyclophosphan  i/v by 250 mg N5                                      3.       platidiam      i/v by 16 mg N5.                                      ______________________________________                                    

The patient endured the treatment well, vomiting occurred within thefirst two days of the cycle.

X-ray Aug. 22, 1991: Favorable dynamics expressed by a decrease in thetumor focus size to 7.5%--9 cm.

Blood analysis Aug. 20, 1991: leucocytes--4,100

Aug. 23, 1991: transferred to the rehabilitation department, receivedcourses of general supporting and symptomatic therapy.

    ______________________________________                                        08.26.91: leucocytes 2,600                                                    Blood analysis 08.30.91:                                                      leucocytes           1,200                                                    hemoglobin           9.0                                                      erythrocytes         2,700                                                    ESR                  10                                                       thrombocytes         20% - 64,000                                             08.30.91: 12.0 ml of NPD-A solution                                           administered subcutaneously.                                                  Blood analysis 09.02.91:                                                      leucocytes           2,800                                                    Blood analysis 09.05.91:                                                      leucocytes           3,200                                                    hemoglobin           10.8                                                     erythrocytes         3,200,000                                                ESR                  3                                                        thrombocytes         25% - 80,000                                             Blood analysis 09.09.91:                                                      leucocytes           7,000                                                    thrombocytes         60% - 138,000                                            hemoglobin           8.5                                                      erythrocytes         2,300,000                                                ______________________________________                                    

The child was discharged for an interval after the treatment course.

EXAMPLE 5

A female child, 11 years old, weight 29.5 kg, height 13 cm was diagnosedas having accessory skull sinuses tumor. She had been operated on priorto admission to the Oncology Center.

    ______________________________________                                                   Treatment:                                                         ______________________________________                                                   07.09.91-07.12.91                                                             08.01.91-08.04.91:                                                 ______________________________________                                    

administered along the following scheme:

    ______________________________________                                                      1 day                                                                              2 day     3 day  4 day                                     ______________________________________                                        vinkristin (mg) i/v                                                                           1.5    0.5       0.5  0.5                                     adriamycin (mg) i/v    30        30                                           cyclophosphan (mg) i/v                                                                        600                                                           platidiam (mg) (i/a)                  60                                      ______________________________________                                    

Lumbar punctures were made twice in the course of treatment: no blasticcells detected.

Aug. 26, 1991: upon control examination in the Polyclinic or Scientificand Research Institute of Pediatric Oncology:

Blood analysis: leucocytes--4,600

Another course of polychemotherapy began:

    ______________________________________                                        vincristine    1.5    mg i/v  08.26.91                                        cyclophosphan  600    mg i/v  08.26.91                                        vincristine    0.5    mg i/v  08.27.91                                        farmarubicin   30     mg i/v  08.27.91                                        Blood analysis 08.28.91:                                                              leucocytes                                                                           1,600                                                          ______________________________________                                    

PCT course aborted; the child was transferred to the rehabilitationdepartment.

Aug. 30, 1991: 15.0 ml of NPD-A solution administered subcutaneously

    ______________________________________                                        Blood analysis 09.02.91:                                                      leucocytes       2,800                                                        Blood analysis 09.04.91:                                                      leucocytes       3,400                                                        hemoglobin       13.2                                                         erythrocytes     4,160,000                                                    ESR              2                                                            thrombocytes     40% - 166,400                                                Blood count:                                                                  basophils        2                                                            eosinophils      6                                                            juvenile neutrophils                                                                           1                                                            stab neutrophils 2                                                            segmented neutrophils                                                                          46                                                           lymphocytes      34                                                           monocytes        9                                                            Blood analysis 09.09.91:                                                      leucocytes       3,500                                                        hemoglobin       11.6                                                         erythrocytes     3,430,000                                                    ESR              3                                                            thrombocytes     45% - 154,350                                                Blood count:                                                                  eosinophils       7                                                           stab neutrophils  2                                                           segmented neutrophils                                                                          39                                                           lymphocytes      38                                                           monocytes        14                                                           ______________________________________                                    

Sep. 10, 1991: the girl was transferred to the NOC Clinic for treatmentcontinuation.

EXAMPLE 6

Male child 5 years old. Weight 18 kg, height 112 cm was diagnosed ashaving acute lymphoblastic leucosis 2.

Treatment

Since May 14, 1990: a PCT course along the following scheme:

    ______________________________________                                        1.      prednisolone 40 mg daily                                              2.      vincristine  i/v, 5 by 1.0 mg                                         3.      methotrexate endolumbarly, 4 by 7.5 mg                                4.      rubomycin    i/v, 1 by 20 mg;                                         ______________________________________                                        Myelogram 05.30.90:                                                           blastic cells    1.0%                                                         Blood analysis 06.18.90:                                                      leucocytes       5100                                                         ESR              2                                                            erythrocytes     3,000,000                                                    hemoglobin       9.4                                                          thrombocytes     240,000                                                      Blood count:                                                                  segmented neutrophils                                                                          64                                                           lymphocytes      33                                                           monocytes         3                                                           ______________________________________                                        Since 07.23.90: a PCT course (remission                                       consolidation) along the following scheme:                                    1.      prednisolone  40 mg daily                                             2.      cytosar       5 by 70 mg                                              3.      L-asparaginase                                                                              6 by 15.000 units                                       4.      methotrexate  3 enlarged doses (300, 700                                                    and 500 mg)                                             ______________________________________                                    

After the PCT course radiation treatment was administered forneuroleucosis prophylactic, total dosage 24 Gray; the patient enduredthe treatment well;

Myelogram Sep. 12, 1990: 1.0% of blastic cells;

Discharged for off-hospital supporting therapy.

Oct. 24, 1991: admitted to the NOC children's rehabilitation departmentfor a PCT course, upon admission--2000 leucocytes in the blood analysis.

Oct. 25, 1991: 12.0 ml of NPD-A solution administered intramuscularly.

    ______________________________________                                        Blood analysis 10.28.91                                                       leucocytes       2700                                                         Blood analysis 10.30.91:                                                      leucocytes       4200                                                         ESR              0                                                            erythrocytes     4,320,000                                                    hemoglobin       12.5                                                         thrombocytes     35% - 150,000                                                Blood count:                                                                  eosinophils       1                                                           stab neutrophils  1                                                           segmented neutrophils                                                                          62                                                           lymphocytes      21                                                           monocytes         16;                                                         ______________________________________                                    

EXAMPLE 7

A female child 13 years old, weight 34 kg, height 157 cm, diagnosed ashaving Ewing's sarcoma.

Treatment

Oct. 19, 1991-Oct. 24, 1991:a PCT course along the following scheme:

    ______________________________________                                                        day 1  day 2     day 3                                                                              day 4                                   ______________________________________                                        vinkristin, mg (i/v)                                                                          1.8                                                           adriamycin, mg (i/v)                                                                          60                                                            cyclophosphan, mg (i/v)                                                                              1400                                                   platidiam, mg (i/a)                   100                                     ______________________________________                                        Blood analysis 10.23.91:                                                      leucocytes       3100                                                         ESR              24                                                           erythrocytes     4,380                                                        hemoglobin       13.2                                                         thrombocytes     80% - 301,000                                                Blood count:                                                                  stab neutrophils  6                                                           segmented neutrophils                                                                          76                                                           lymphocytes      14                                                           monocytes          4;                                                         ______________________________________                                    

Oct. 28, 1991: transferred to the children's rehabilitation department.

    ______________________________________                                        Blood analysis 10.31.91:                                                      ______________________________________                                        leucocytes     1200                                                           erythrocytes   11                                                             hemoglobin     13.2                                                           thrombocytes   60% - 288,000                                                  Blood count:                                                                  segmented      5                                                              lymphocytes    14                                                             monocytes      6                                                                             (calculated for                                                               25 cells);                                                     ______________________________________                                    

Oct. 31, 1991: 15.0 ml of NPD-A solution as administeredintramuscularly.

    ______________________________________                                        Blood analysis 11.02.91:                                                      leucocytes       1700;                                                        Blood analysis 11.04.91:                                                      leucocytes       3100                                                         ESR              5                                                            erythrocytes     4,880                                                        hemoglobin       14.1                                                         thrombocytes     50% - 244,000                                                Blood count:                                                                  basophils         1                                                           eosinophils       3                                                           stab neutrophils  2                                                           segmented neutrophils                                                                          13                                                           segmented neutrophils                                                                          61                                                           monocytes         20;                                                         Blood analysis 11.12.91:                                                      leucocytes       5000;                                                        ______________________________________                                    

Nov. 13, 1991: transferred to the NOC Pediatric Oncology Department fortreatment continuation.

EXAMPLE 8

A female child, 15 years old, weight 56 kg, height 169 cm was diagnosedas having lymphogranulomatosis, primary lesion ofservico-supraclavicular lymph nodes on both sides and mediastinum. Shehad 5 previous PCT courses administered (adriamycin included in thethird one) at the National Oncology Center.

Treatment

Since Jan. 1, 1991 the girl was transferred in the children'srehabilitation department where she received PCT along the followingscheme:

    __________________________________________________________________________           d.1                                                                              d.2                                                                              d.3                                                                              d.4                                                                              d.5                                                                              d.6                                                                              d.7                                                                              d.8                                                                              d.9                                                                              d.10                                                                             d.11-14                                                                           d.15                                 __________________________________________________________________________    prednisalon,                                                                         60 mg daily throughout the whole cycle                                 mg (orally)                                                                   cyclophosphan,                                                                       950                  950                                               mg (i/v)                                                                      vinblastin                                                                           9.5                  9.5                                               mg (i/v)                                                                      natulan,                                                                             50 100                                                                              150                                                                              150                                                                              150                                                                              150                                                                              150                                                                              150                                                                              150                                                                              150                                                                              by 200                                   mg (orally)                                                                   pharmarubycin,              60           60                                   mg (i/v)                                                                      __________________________________________________________________________    Blood analysis 10.08.91:                                                      leucocytes             3300                                                   Blood analysis 10.11.91:                                                      leucocytes             3500                                                   ESR                    2                                                      erythrocytes           3,630                                                  hemoglobin             14.2                                                   thrombocytes           70% - 252,000                                          Blood analysis 10.15.91:                                                      leucocytes             1500                                                   __________________________________________________________________________

Oct. 15, 1991: 12.0 ml of NPD-A solution administered intramuscularly.

    ______________________________________                                        Blood analysis 10.16.91:                                                      leucocytes       1800                                                         Blood analysis 10.18.91                                                       leucocytes       3700                                                         Blood analysis 10.25.91:                                                      leucocytes       7200                                                         ESR              3                                                            erythrocytes     3.760                                                        hemoglobin       12.5                                                         thrombocytes     70%; 259,000                                                 Blood count:                                                                  stab neutrophils  1                                                           segmented neutrophils                                                                          86                                                           lymphocytes       6                                                           monocytes          7;                                                         ______________________________________                                    

The girl was discharged in a satisfactory state, consultation in the NOCclinic was recommended in two months.

EXAMPLE 9

Intensive chemo and radiation therapy are presently used for treatinghemoblastoses and malignant tumors. Such treatment generally results inlesions of the gastroenteric tract mucosal membranes found in 70-90% ofthe patients. This leads to problems of feeding up to complete anorexia,adds to the suffering of the patient and produces a noticeable negativeinfluence upon the treatment results. The main disease process iscomplicated in this case by disbacteriosis, medication-inducedstomatitis and secondary infections. Generally accepted methods ofarresting emerging complications (oxicort, antiseptic solutions,antibiotic solutions, vitamins, antivirals, etc.) are, as a rule,ineffective.

0.25% NPD solution in 0.1% NaCl in 10 and 20 ml bottles was used fortreatment.

481 patients suffering from lesions of the gastroenteric tract mucosalmembranes resulting from extensive chemo and/or radiation therapy wastreated in the following manner. All food fragments were removed fromthe mouth cavity. Each patient then rinsed their mouth cavity with 3-5ml of a 0.25% DNA-sodium solution in 0.1% NaCl for 3-5 minutes withgradual swallowing of the solution with the saliva. The procedure wasrepeated from 6 to 10 times, depending on the graveness of the patient'sstate and local symptoms. Treatment was repeated as needed for up to oneweek. All patients regardless of the seriousness of the lesion showed apronounced positive effect by the 2nd-4th day. Those having mouth cavitymucosal membrane expanded lesions exhibited a clear membrane almostcompletely by the 2nd day. In case of ulceronecrotic lesions, thepatients allowed their mouth cavity to be examined in detail, wereallowed to chew and swallow soft food, the mucosal membrane was fullycleared and epithelized on the 3rd-4th day. Those patients exhibitingcolitis of the mouth cavity showed similar positive results.

In another embodiment of the present invention, various metal complexeswere prepared with the DNA-sodium salt obtained from sturgeon milt andfound to have high antiviral activity, and are therefore useful for thetreatment of diseases particularly those caused by the HIV virus. Thecompounds which are particularly useful in the practice of thisembodiment of the invention are the DNA-sodium salts which have beencomplexed with certain polyvalent metals. Suitable metals which can formcomplexes with the DNA-sodium salts of the present invention includemagnesium, calcium, cobalt, nickel iron, zinc, selenium and gold. Themetal is used in the form of water-soluble compound such as salts andbases. The complexes are obtained by mixing the DNA-sodium salt and themetal compounds dissolved in water at a temperature of from about 5 toabout 40° C. and for a period of from about 5 minutes to about 2 hours.The preferred metals are zinc, nickel, cobalt and iron. The complexescan be prepared over a wide range of molar proportions of the DNA-sodiumsalt or polyvalent metal.

For example, the molar ratio of DNA-sodium to metal can range between1:1 to 1000:1. These complexes of the DNA-sodium salt and the polyvalentmetal have been given the designation AC and are identified as follows:

AC-1 for the Zm metal complex with Zn

AC-2 for the Ni metal complex with Ni

AC-3 for the Co metal complex with Co

AC-4 for the Fe metal complex with Fe

As indicated, the metal complexes were found to possess excellentantiviral properties.

The acquired human immunodeficiency syndrome (AIDS) has obtained worldattention and much effort is being expended to inhibit the spread ofthis disease ad also to treat those who have become afflicted. Wheninfected with the HIV virus, helper T-cells as well as others aredestroyed leading to serious opportunistic infections and eventuallydeath. No effective therapy for AIDS has been established except AZTwhich is a reverse transcriptase inhibitor and does provide someimprovement in clinical symptoms. However, AZT is relatively toxic andin many instances causes adverse reactions.

Today AZT is known as the most popular antiviral preparation revealingin vitro high activity against retroviruses, HIV among them. AZT is usedfor supporting therapy at early stages of HIV infecting as well as thedeep stage of HIV illness, AIDS. This preparation as indicated, ischaracterized by insufficient efficacy and high toxicity. AZT fails tocompletely stop the development of HIV in in vitro systems while optimaldoes of this preparation kill about 30% of normal cells. For cells, theAZT LD₅₀ does not exceed 0.1 M, 10 mg/kg of AZT injected intercerebrallyto mice causes death of 50% of animals.

The objective of this embodiment of the invention was to create a lesstoxic antiviral agent revealing a stronger antiviral effect against HIV.This objective was achieved by producing a highly active preparation bycomplexing a DNA sodium salt (DNA-NA) with polyvalent metals aspreviously indicated. The complex, when introduced into the organism,penetrates, by means of endocytosis, into the cytoplasm of an activelyfissing cell, lymphocyte, for example, binding both the reversetranscriptase (by forming connections through the metals constitutingthe complex) and HIV proteins produced by the infected cell genome(through interaction with the DNA part of the complex). The new stabletriple complexes are, under the influence of intracellular ferments,gradually destroyed into separate fragments lacking anti-HIV activeness,and then utilized. As a result of these processes the development of HIVin the cell is inhibited by practically 100%. Thus, the characteristicfeature of the antiviral preparation is its high anti-HIV activeness andlow toxicity.

EXAMPLE 10

Complexes of DNA-Na with zinc, cobalt, nickel and iron were used for HIVsuppression in in vitro systems with various concentrations of thecomponents in the complexes. AZT manufactured by "Wellcome" Company wasused for comparison purposes. The antiviral activity of the preparationwas tested according to the method recommended by WHPO for thesepurposes. CEM-SS and MT-4 reinoculated human cell lines were used. Thecells were cultivated in the concentration of (0.03-0.05)*10⁶ cells per1 ml of RPMI 1640 medium with 10% calf fetal serum, 300 mg/ml ofL-glutamine, 100 mcg/ml of gentamicin and grown in the form of asuspension. The vitality of cells was tested by dyeing them with 0.4%solution of threpane blue dye stuff. HIV-IVS and HIV-1 HTLV/IIIB strainswere used as the virus sources.

The cell suspension was placed into 24 well panels, processed by variousdoses of preparations and then infected by HIV. The infectionmultiplicity was 0.01 TCD₅₀ per cell. After that, the cultures wereincubated at 37° C. in a 5% CO₂ atmosphere and 98% humidity for 5-7 daysup to the moment of defining the cytopathic effect of the virus upon thecell culture.

Immunophermentative analysis was used to define formation of viralantigen.

The results are listed in Table 1.

    __________________________________________________________________________    ANTIVIRAL EFFECTIVE AND CYTOTOXICITY OF PREPARATIONS                                                        CYTOTOXICITY                                                   QUANTITY           QUANTITY OF                                          DNA - Na                                                                            OF SYNCITIA IN                                                                          IFA      CELLS IN ml OF                                                                          VITALITY                                   Me    % FROM THE CON-                                                                         OPTICAL  CULTURAL  OF                                PREPARATION                                                                            M/M   TROL VIRUS                                                                              DENSITY                                                                            ng/ml                                                                             LIQUID × 10.sup.8                                                                 CELLS %                           __________________________________________________________________________    DNA - Na 4:9   --        --   --  --        --                                Zn       3:10  25        --    0.25                                                                             0.83      69                                         0.5:350                                                                             35        --    0.50                                                                             0.88      81.2                                       1:1000                                                                              70        0.487                                                                              --  0.86      84.3                                       1:1100                                                                              100       0.597                                                                              --  0.89      86.2                              DNA - Na 4.9   --        --   --  --        --                                Ni       3:10   0        --    0.1                                                                              0.82      71.0                                       0.5:350                                                                              0        --   >2.0                                                                              0.86      79.0                                       1:1000                                                                              68        --   >2.0                                                                              0.85      84.3                                       1:1100                                                                              100       0.607                                                                              --  0.88      87.5                              DNA - Na 4:10  --        --   --  --        --                                Co       3:10  10        0.190                                                                              --  0.86      78.4                                       0.5:350                                                                             15        0.184                                                                              --  0.84      87.1                                       1:1000                                                                              66        0.414                                                                              --  0.89      76.3                                       1:1100                                                                              100       0.585                                                                              --  0.87      82.1                              DNA - Fe 3:9   --        --   --  --        --                                Fe       3:10   0        0.103                                                                              --  0.87      79.3                                       0.5:350                                                                              0        0.247                                                                              --  0.84      82.1                                       1:1000                                                                              61        0.492                                                                              --  0.87      81.7                                       1:1100                                                                              100       0.595                                                                              --  0.85      84.5                              A T             8        0.091                                                                               0.1                                                                              0.61      55.2                              CONTROL VIRUS  100       0.602                                                                              >2.0                                                                              --        --                                CONTROL CELLS   0        0.087                                                                              <0.1                                                                              0.91      86.3                              __________________________________________________________________________

As is evident from the above table, DNA-Na complexes with zinc, cobalt,nickel and iron in a diverse spectrum of DNA/Me percentages (M/M) andexhibit high anti-HIV activeness while practically not revealing anycytotoxicity, an advantage over AZT.

In a further evaluation of the metal complexes of the DNA-sodium salt,applicants have evaluated preparations AC-1 and AC-4, obtained by themethods of this invention and found them to be highly active againstAIDS in tests on cells and less toxic than "Retrovir" in tests on smalllaboratory animals. It is evident from the following methods used toevaluate the materials and the data set forth in Tables 2 and 3, thatthese materials have unexpected properties which render them useful inthe treatment of AIDS.

EXAMPLE 11

Antiviral activity of these preparations toward HIV-1 was studied usingas the model, cellular cultures infected with HIV-1.

The testing of anti-virus activity was conducted using two types ofinterwoven cellar lines sensitive to infecting by AIDS-virus--the MT-4cells and Yurkat tat III cells. The cells were harvested in suspensionform in a PPMI 1640 medium containing 15% of fetal serum with exposureto the atmosphere characterized by 5% CO₂ content, 98% humidity leveland 37% C. temperature level. The cells were infected by the HTLV-IIIBculture of AIDS-1 virus in accordance with Who recommendations forconducting scientific researches on testing pharmaceutical preparations.The methods used for determining anti-virus activity of the testedpreparations are recommended for similar purposes by the WHO ReferenceLaboratory (Belgium) headed by Professor de Clerk who is aninternationally recognized leading expert in synthesis and screeningchemical preparations for AIDs treatment (I. Antimicrob. Chemoter.,1989, No. 23, Suppl. A 35-46). The work was done at the VirologyInstitute of the U.S.S.R. Academy of Medical Sciences on MT-4 humanlymphoid cells and on Yurkat tat III cells as indicated above.

The cells were incubated for 6 days under similar conditions andthereafter their viability was determined as well as formation ofvirus-induced syncytia and virus antigen (p24) using immunofermentalanalysis. The formation of syncytia, huge cell conglomerates, is one ofthe HIV reproduction characteristics. The formation of the syncytia isconnected with the interaction of gp120 virus protein, situated on themembranes of the infected cells, and CD4 receptor, situated on themembranes of non-infected cells. As a result of this interaction, themerging of nuclear membranes occurs and a cell culture lacking immuneability, consisting of many cells nuclei, is formed. The viable cellswere counted using thripane blue dye-stuff in the Goryayev chamber. Theerror of the method in all cases does not exceed 5%.

The studies on toxicity of AC-1 and AC-4 and of aziodothymidine wereconducted on non-linear white mice weighing 6 to 7 grams, making use ofdifferent concentrations of preparations per kg of weight of the animalin the volume of 0.2 ml in hypodermic, intranasal and intraabdominalinjections and 0.03 ml in intracerebral injections. The animals werekept under observation for 2 weeks and then LD₅₀ was calculated usingCurber method. The lethal dose of the preparation which has caused deathof 50% of the animals was considered an LD₅₀ dose. The preparation wasused as a sterile solution was a concentration of 0.05%.

Results: The results of the studies in anti-virus activities of thesepreparations are presented in Table 2 below:

                  TABLE 2                                                         ______________________________________                                        Determining anti-virus activity of                                            AC-series preparations on cells                                               (on the 6th day)                                                              preparation                                                                           preparation                                                                              number of                                                                              immunofermental                                                                          viability                              code    dose mkg/ml                                                                              syncitia %                                                                             activity ng/ml                                                                           %                                      ______________________________________                                        AC-1    100        75       2.0        10                                             250        75       2.0        10                                             350        25       0.25       69                                     AC-4    100        none     1.35       21                                             250        none     0.5        32                                             350        none     0.1        71                                     AZT     270        none     0.1        96                                     ______________________________________                                    

As evident from Table 2 above, the preparation AC-1, essentially, andthe preparation AC-4, fully (in a dose of 350 mkg/ml) inhibits growth ofHIV-1 virus similar to azidothymidine. Additionally, the AC-4preparation does not exhibit toxic effect upon cells.

Studies on Toxicity of Preparations on the Model of Laboratory Animals(White Mice)

Tests were conducted in parallel with 4 codified preparations incomparison with the preparation azidothymidine in doses of 5, 10, 20, 35and 50 mg/kg. The studies on toxicity of the four preparation, namelyAC-1, AC-2, AC-3, AC-4 and azidothymidine have been conducted onnonlinear white mice weighing 6 to 7 grams, making use of differentconcentrations of preparations per kg of weight of the animal in thevolume of 0.2 ml in hypodermic, intranasal and intraabdominal injectionsand 0.03 ml in intracerebral injections. The animals were kept underobservation for 2 weeks and then LD₅₀ was calculated using Curbermethod. The lethal dose of the preparation which caused death of 50% ofthe animal was considered the LD₅₀ dose.

The results of toxicity studies for these preparations are shown inTable 3.

                  TABLE 3                                                         ______________________________________                                        Estimating toxicality of preparations on white mice.                                         Method     Preparation dose                                    NN   Preparation                                                                             of         mg/kg                                               nn   code      introduction                                                                             5   10   20  35   50  75                            ______________________________________                                        1.   AC-1      in/cerebr. 6/6 6/6  6/6 6/6  6/6 6/6                                          hypoderm.  6/6 6/6  6/6 6/6  6/6 6/6                                          in/abdom.  6/6 6/6  6/6 6/6  6/6 6/6                                          in/nasal   6/6 6/6  6/6 6/6  6/6 6/6                           2.   AC-2      in/cerebr. 6/6 6/6  6/6 6/6  6/6 6/6                                          hypoderm.  6/6 6/6  6/6 6/6  6/6 6/6                                          in/abdom.  6/6 6/6  6/6 6/6  6/6 6/6                                          in/nasal   6/6 6/6  6/6 6/6  6/6 6/6                           3.   AC-3      in/cerebr. 6/6 6/6  6/6 6/6  6/6 6/6                                          hypoderm.  6/6 6/6  6/6 6/6  6/6 6/6                                          in/abdom.  6/6 6/6  6/6 6/6  6/6 6/6                                          in/nasal   6/6 6/6  6/6 6/6  6/6 6/6                           4.   AC-4      in/cerebr. 6/6 6/6  6/6 6/6  6/6 6/6                                          hypoderm.  6/6 6/6  6/6 6/6  6/6 6/6                                          in/abdom.  6/6 6/6  6/6 6/6  6/6 6/6                                          in/nasal   6/6 6/6  6/6 6/6  6/6 6/6                           5.   AZT (azido                                                                              in/cerebr. 6/6 6/6  6/6 6/6  6/6 6/6                                thymidine)                                                                              hypoderm.  6/6 6/6  6/6 6/6  6/6 6/6                                          in/abdom.  6/6 6/6  6/6 6/6  6/6 6/6                                          in/nasal   6/6 6/6  6/6 6/6  6/6 6/6                           6.   Placebo   in/cerebr. 6/6 6/6  6/6 6/6  6/6 6/6                                          hypoderm.  6/6 6/6  6/6 6/6  6/6 6/6                                          in/abdom.  6/6 6/6  6/6 6/6  6/6 6/6                                          in/nasal   6/6 6/6  6/6 6/6  6/6 6/6                           ______________________________________                                         NOTES:                                                                        numerator  the number of surviving mice                                       denominator  the number of infected mice.                                

It is evident from the Tables that all of the 5 preparations, no matterwhat their doses were, provided non-toxic for 6 to 7-gram white mice inhypodermic, intranasal and intraabodominal injections (2 weeksobservation). However, 50% lethality in white mice has been registeredwhen increasing 50 mg/kg dose of AC-4 preparation in intracerebralinjections. The preparation azidothymidine also caused a 50% lethal ratein white mice when used in intracerebral injections in doses of 10 to 50mg/kg per weight of the animal. So, the toxic effect of theazidothymidine preparation has been established for 6 to 7 gram whitemice in intracerebral injections as LD50 dose--10 mg/kg of weight.

The toxic effect of an AC-4 preparation using intracerebral injection of5 mice has been recorded at the dose of 75 mg/kg of weight (LD50 dose).

a) All the titres of the AC-series preparations proved non-toxic for 6to 7 gram white mice in all injection methods at doses of 5 to 50 mg/kgof weight.

b) Azidothymidine was also non-toxic for white mice in all injectionmethods at doses of 5 to 50 mg/kg of weight except in intracerebralinjections in doses of 10 to 50 mg/kg of weight.

c) Although the tested preparations showed lower anti-virus activity incomparison with azidothymidine, nevertheless, AC-4 in a dose of 350mkg/ml shows good anti-virus activity and no toxic effect on the testedcalls. Hence, the LD₅₀ of AC-4 is at least 5 times that of AZT.

In summary, it was observed that:

1. Azidothymidine (AZT) in a dose of 1.0 mcm/ml completely suppressesthe AIDS-1 virus development (virus-induced syncitia absent), theantigen (p24 content, as determined by immunofermental analysistechnique) decreased from 2.0 ng/ml virus control down to 1 ng/ml.

2. AC-1 preparation in the same dose of 1.0 mcm/ml almost completelysuppresses the AIDS-1 virus development (the amount of syncytiadecreases from 100% of virus control to less than 25% of testsuspension), the antigen content decreases from 2.0 ng/ml down to 0.25ng/ml. Increasing the AC-1 preparation dose to 10.5 mcm/ml leads to acomplete suppression of the AIDS-1 virus development according to thevirus TITR test.

3. AC-4 preparation in a dose of 1.0 mcm/ml completely suppresses theAIDs-1 virus development (no syncytia), the antigen content decreasesfrom 2.0 ng/ml down to 0.1 ng/ml.

4. On the cell level, all the above biologically active concentrationsare nontoxic for AZT and AC-1 and AC-4 preparations as well.

5. In tests with animal in case of intramedullary injection of AZT innonlinear white mice in a dose of 10 mg/kg of weight, 50% of animalsdie. At the same time, intramedullary injection of AC-1 and AC-4preparations in a dose of 50 mg/kg (5 times as high) produced no lethaleffect on animals.

Thus, AC-1 and AC-4 preparations are at least five times less toxic thanAZT.

Since the bioactive materials of the present invention are much lesstoxic than AZT, they can be utilized in larger doses for the inhibitionof HIV. For example, while AZT is employed for oral administration incapsule form containing 100 mg per unit dose, larger amounts of thematerial of the present invention can be used in single dose forms fororal administration in amounts up to 500 mg and higher, and preferably50 to 500 mg.

As previously indicated, the pharmaceutical compositions containing thebioactive material and a pharmaceutically acceptable carrier can beformulated by methods known in the art. A wide variety of carriers canbe used and include, but are not limited to, inert materials such ascornstarch, magnesium stearate, microcrystalline, cellulose, sodiumstarch glycolate and the like. The concentration of the bioactivematerial in the pharmaceutical compositions can vary depending upon thedosage required, frequency of administration and the age, weight andcondition of the patient or host to whom the composition isadministered. If desired, the pharmaceutical compositions can containone or more other ingredients which are active against the AIDS virusand include compounds such as AZT, DDI and the like.

As indicated above, the bioactive materials of the present invention areuseful for inhibiting the growth of viruses which are susceptible totreatment by such materials. The bioactive materials of the presentinvention inhibit the interaction of gp 120 virus protein with CD4receptor on cellular surfaces of a host and hence are useful forinhibiting the growth of the AIDS virus.

In another embodiment of the invention, compositions containing theDNA-sodium salt have application in the cosmetic field. The DNA-sodiumsalt compositions have been successfully used as components in creamsand lotions. The compositions are useful for preventing agingpigmentation of the skin, removing blackheads, pimples, scars, healingcuts and burns, reducing inflammation and acting as a rejuvenating creamto make the skin feel soft and elastic. It has also been observed thatthe DNA-sodium salt is useful for strengthening and restoring hair. Itcan also be used as a mouthwash or in toothpaste since it has a strongantiperiodontosis effect. The DNA-sodium salt can be used informulations for the aforementioned uses, as a powder, solution or ingel form.

A typical cosmetic preparations containing NPD-A was prepared forsmoothing aging skin. The data showing the results on the generalpharmacological activeness and toxicological characteristics are set outin Examples 12 and 13 below.

The composition is prepared for topical application having from about0.05 to about 10.0 per cent of the active ingredient and preferably fromabout 0.3 to about 0.8 per cent by weight of the cosmetic preparation.

EXAMPLE 12

A cosmetic preparation for topical application for the treatment ofaging skin was prepared by the following materials in % by weight:

    ______________________________________                                        DNA - sodium      0.36                                                        rose water        3.5                                                         C.sub.15 -C.sub.20 hydrocarbons                                                                 2.0                                                         dehydrated lanoline                                                                             1.0                                                         vodlan-60         1.0                                                         glycerin monostearate                                                                           2.0                                                         emulsion waxes    2.0                                                         distilled monoglycerides                                                                        2.0                                                         butylstearate     5.0                                                         sodium laurylsulfate                                                                            0.4                                                         olive oil         10.0                                                        perfume oil       6.0                                                         paraoxybenzol acid methyl                                                                       0.5                                                         ether                                                                         fragrance         0.6                                                         balance           distilled water                                             ______________________________________                                    

EXAMPLE 13

The cosmetic composition prepared in Example 12 was administered to 15women varying in age from 30 to 56 years. The composition was applied tothe face and hand skin 2 times daily for 21 days by rubbing into theskin with circular hand movements.

The resilience of the skin was studied among 8 of the test subjects withthe help of an experimental device with a piezoelectric sensitiveelement designed in the Institute of Biophysics of Russian Academy ofSciences. After the first application, skin resilience was measured insymmetric points of the face skin (above the eyebrow, on the cheekbone,near the corner of the mouth, and under the lower jaw) before and 5 to10 minutes after the application. The composition rapidly andeffectively entered the skin after its application and the averagevalues of skin resilience after the application remained practicallyunchanged.

All of the patients noted the sensation of skin "freshness" during thecourse of treatment and by the time it was finished, face skin becamesmooth and its nutrition was promoted. Average skin elasticity enlargedby 15-20%.

There was no evidence of skin irritation, allergic reactions or anyother side effects on any of the patients.

In addition to the use of the DNA-sodium salts in formulations for thetreatment of skim for cosmetic purposes, they are also useful, aspreviously indicated, formulations for their grooming, toothpaste,mouthwash and the like. In such formulations the DNA-sodium salt can bepresent in concentrations of from about 0.1 to about 1.0 per cent byweight of the formulation. The DNA-sodium salts can be used, as the soleactive ingredient in such formulations or in combination with otheractive components.

The formulation base for of the various products is comprised ofingredients known in the art for such applications the only requirementbeing that none of the ingredients contained therein, adversely affect,or are adversely affected by the DNA-sodium salt.

Although the invention has been illustrated by the preceding examples,it is not to be construed as being limited to the materials employedtherein, but rather, the invention is directed to the generic area aherein after disclosed. Various modifications and embodiments thereofcan be made without departing from the spirit or scope thereof.

What is claimed is:
 1. A DNA-sodium salt-metal complex comprising:1) ADNA-sodium salt composition comprising:a) not less than 80 percent byweight of DNA-sodium salt extracted from sturgeon milt wherein thehyperchromicity effect (G) is not less than 37 percent; b) not more than1 percent protein by weight, c) not more than 17 percent moisture byweight, and 2) a polyvalent metal.
 2. The complex of claim 1 wherein thepolyvalent metal is selected from the group consisting of zinc, cobalt,nickel and iron.
 3. The complex of claim 1 wherein the polyvalent metalis zinc.
 4. The complex of claim 1 wherein the polyvalent metal iscobalt.
 5. The complex of claim 1 wherein the polyvalent metal isnickel.
 6. The complex of claim 1 wherein the polyvalent metal is iron.